A design principle for a single-stranded RNA genome that replicates with less double-strand formation

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

We investigated the effect of the insertion of loop structures. We first used RNAs with insert sequences that form 4-to 20-nt loop structures consisting of G and A (1. Loop GA4, 2. Loop GA6, 3. Loop GA8, 4. Loop GA10 and 5. Loop GA20). We incubated these RNAs (100 nM) with Q␤ replicase (100 nM) and nucleotides containing [ 32 P]-UTP for 25 min at 37 • C and measured the ssRNAs and dsRNAs in the replication product by autoradiography after separation by agarose gel electrophoresis. We plotted the sum of the newly synthesized ssRNA and dsRNA amounts (total RNA synthesis) and the ratio of newly synthesized dsRNA to the newly synthesized total RNA (dsRNA ratio). Depending on the size of the inserted loop, total RNA synthesis exhibited a tendency to decrease ( Figure 3A ). The dsRNA ratio decreased by the insertion of 4-to 8-nt loops but increased by the insertion more than 8-nt loops ( Figure 3B ). This increase of the dsRNA ratio by larger loops supports the notion that dsRNA formation is mediated by loop regions in a template RNAs and is consistent with the prediction according to the model shown in Figure 1 .